非酒精性脂肪性肝病中MAIT细胞的免疫学特性变化及活化调控机制研究

Nonalcoholic fatty liver disease (NAFLD) is one of the most common chronic liver diseases worldwide. The immune response contributes critically to NAFLD progression. Mucosal-associated invariant T (MAIT) cells are a recently identified population of innate-like lymphocytes and are abundant in the mucosa and liver. MAIT cells can recognize ligands which are presented by MHCI-related molecule. Our previous study found the frequency of circulating MAIT cells was significantly lower in NAFLD patients compared to healthy controls, while the number of MAIT cells increased in livers of NAFLD patients. At the same time, MR1 mainly expressed on Kupffer cells in NAFLD liver. Upon MCD feeding, MR1-/- mice displayed more obvious steatohepatitis than that of wild type mice, suggesting that MAIT cells play an important role in the progression of NAFLD. Firstly, this project intends to explore the changes in the immunological properties of intrahepatic MAIT cells during different stages NAFLD (lipid deposition-inflammation-fibrosis) through animal experiments. Secondly, regulation mechanism of the MAIT cells’ activation will be studyed by free fatty acid/lipopolysaccharide-stimulated-macrophage experiments in vitro. In additon, we will analyze the immunological effects of MAIT cells in different ways of activation. Our research will further reveal the immunological pathogenesis of NAFLD and provide a scientific basis for the development of new immunotherapeutic strategies.

非酒精性脂肪性肝病（NAFLD）已为慢性肝病的最常见病因之一，免疫反应在其发生发展中起重要作用，黏膜相关恒定T（MAIT）细胞是一群在黏膜及肝脏表达丰富的新型天然免疫细胞，可识别由MR1分子递呈的配体。我们前期工作发现，MAIT细胞在NAFLD患者外周血中频率降低，而在肝脏组织内数量增多，同时MR1分子在肝脏Kupffer细胞表面表达升高，MR1-/-小鼠（即MAIT细胞缺乏）脂肪肝模型较野生型小鼠炎症加重，提示MAIT细胞在NAFLD发展中发挥重要作用。本项目拟通过动物实验探讨NAFLD发生发展过程中（脂质沉积-炎症-纤维化不同时期）MAIT细胞的免疫学特性的变化，以及体外游离脂肪酸/脂多糖刺激巨噬细胞实验探索MAIT细胞的活化调控机制，并分析MAIT细胞在不同方式活化下所发挥的免疫学效应，进一步揭示NAFLD的免疫学发病机制，为研发新的免疫治疗策略提供科学依据。

目的：本项目主要研究黏膜相关恒定T（MAIT）细胞在非酒精性脂肪性肝病（NAFLD）发生发展中的作用机制。.方法：通过小鼠高脂饮食（HFD）及胆碱蛋氨酸缺乏饮食（MCD）建立NAFLD模型，流式细胞术检测野生型（WT）小鼠NAFLD模型肝脏、脾脏、肠道MAIT细胞的频率和功能变化，比较MAIT细胞缺乏的小鼠（MR1-/-）NAFLD模型后表型的变化，并进一步通过免疫荧光和免疫组化等探讨肠道MAIT细胞的激活方式和功能。.结果：野生型小鼠HFD和MCD模型肝脏组织内MAIT细胞频率均增加，与正常饮食小鼠比，MCD小鼠肝脏内IL-4+和IL-10+MAIT细胞频率升高，IFN-γ+频率降低。尤其是HFD模型小鼠结肠固有层MAIT细胞频率明显升高。MR1-/-小鼠HFD模型中其肠道黏膜紧密连接蛋白zo-1及闭合蛋白表达缺失，同时发现Lcn2升高，同时发现MAIT细胞中中TCR激活通路中的特异信号Nur77明显升高，而肠道CX3CR1+吞噬细胞MR1表达增高，提示MAIT细胞通过该CX3CR1+吞噬细胞提呈抗原并依赖TCR方式激活。我们使用16S rRNA基因测序检测野生型小鼠及MR1-/-小鼠在正常饮食及高脂饮食下粪便菌群变化，在门的水平，MR1-/-HFD组小鼠Proteobacteria减少，而Bacteroidetes增多，提示肠道菌群可能在其中其重要作用，目前分析肠道细菌代谢物进一步明确MAIT细胞的激活分子。.结论： 在NAFLD发生发展中，肝脏MAIT细胞分泌细胞因子向Th2型转化，肠道MAIT细胞可被肠道菌群及代谢物激活和扩增，为肠道提供黏膜屏障。此研究为NAFLD的治疗提供新的靶点方向。